Cancer is the second leading cause of death in the United States. There is an ever-growing need for accurate analysis of oncogenic markers for the diagnosis and prognosis of cancer. For example, detection of an array of oncogenic markers may allow physicians to detect early stage cancer and to monitor cancer progression. With knowledge of a patient's responsiveness to anticancer therapies prior to drug initiation, physicians could select the best course of treatment for each individual patient. Furthermore, routine analysis of drug effectiveness during the course of treatment may reveal a patient's unresponsiveness to specific anti-cancer drugs. This information could be used to improve the selection of drug treatment regimens.
Current methods for oncogenic marker analysis are based on interrogating malignant cells in a heterogeneous mixture of normal and cancer cells such as whole blood. Methods such as the LeukoLOCK Total RNA Isolation System (Ambion) capture circulating malignant cells from whole blood by passing a blood sample through disposable leukocyte depletion filters. Typically, these depletion filters are flushed with buffers such as PBS as the malignant cells are recovered. This wash step changes the intracellular concentration of anticancer drugs previously exposed to the cells, thereby possibly causing de novo signaling responses within the cells and altering expression of oncogenic markers. Consequently, expression of oncogenic markers in the analyzed sample could inaccurately reflect a patient's response to specific anticancer therapies. This could lead to incorrect diagnostic and/or prognostic evaluations. The present invention overcomes this potential source of error by providing methods and apparatuses for isolating a subset of blood cells without changing the intracellular concentration of an anticancer drug.